Antimicrobial plastics

Abstract

The bioplastic additives and antimicrobial plastics include polyphenols and organic polymers. The polyphenol can be a phenol, a polyphenol, a bioflavonoid, a flavonoid, a tannin, a coumarin, a lagman, a quinone, a stilbene, or a qurcuminoidenin. The organic polymer may be chitosan, chitin, cellulose, and keratin. In an advantageous form, the polyphenol is hesperidin, and the organic biopolymer is chitosan.

Description

Technical Field

[0001] The present invention relates to plastics or polymers, in particular antimicrobial plastics or polymers. Background Art

[0002] Various surfaces and the materials comprising them are potential breeding grounds for microorganisms such as bacteria and fungi. In addition, various materials and surfaces may prove suitable for supporting viruses.

[0003] The global SARS-CoV-2 pandemic has created a significant demand for new technologies and products with antimicrobial properties. Since many consumer products contain some form of plastic or plastic materials, the addition of plastic additives with antimicrobial properties would be beneficial, including additives that target infectious viral strains such as rhinovirus, influenza, SARS, SARS-CoV-2 (i.e., COVID-19) and dangerous bacterial strains such as Escherichia coli (E. coli), Staphylococcal, Methicillin-resistant Staphylococcus aureus (MRSA), and Methicillin-susceptiable Staphylococcus aureus (MSSA).

[0004] Recently, antimicrobial additives or agents (including antiviral additives) have been incorporated into different materials (including plastics). However, the effectiveness of previous antimicrobial agents (including antiviral additives for plastics) has been limited. For example, typical antimicrobial additives used today are metals (such as zinc and ionic silver, thiazoles and halides). Although these additives are effective, they also have negative effects due to their toxicity in the environment. Especially due to the current SARS-CoV-2 crisis, there are great concerns about the overuse of these materials and the subsequent dangerous accumulation in the environment, with serious long-term exposure consequences.

[0005] There has been a notable lack of innovation in the field of antimicrobial plastics, polymers, and plastic surfaces. For example, the most widely used antimicrobial plastic additives to date are metals such as silver, zinc, and copper. The only recent advances have been simple combinations of these metals with themselves, other ions, or non-reactive compounds. This can be illustrated by the following examples: silver / zinc / copper, silver / citrate, silver sulfadiazine, silver phosphate, silver ceramics, silver palladium, silver platinum, silver zeolites, silver copper, silver / glass, and so on. These combinations are often repeated with zinc or copper metal ions replacing the silver.

[0006] Another current limitation in the field of bioplastics (e.g., antimicrobial plastics) is the method of delivering the antimicrobial agent. Current antimicrobial applications rely on integrated migration. In this method, the additive is mixed within the plastic material and, due to the inherent incompatibility gradient between the antimicrobial additive and the polymer matrix, the additive simply migrates or diffuses toward the surface of the material. This technique and mode of action is not only inefficient but also creates weaknesses and structural instabilities in the plastic material itself due to mechanical instabilities of the structural aggregate.

[0007] Another limitation in the bioplastics field is toxicity due to inherent acute and chronic toxicity. The primary toxicity concern with current state-of-the-art technologies is not specific to humans, but rather focuses on the impacts of landfill runoff on aquatic systems and the potential for damage to marine systems. Current technologies; silver, zinc, copper, arsenic, triclosan, thiazole, arsenic, and oxybisphenoxathane A (OBPA) have demonstrated significant marine toxicity potential.

[0008] Yet another limitation in this field is thermal degradation. Most antimicrobial compounds used today have thermal decomposition constants close to those of the matrix polymer itself. This is a significant disadvantage because as the temperature of the bioplastic increases, it also increases the mobility of the additive and affects or increases its migration rate. This factor significantly impacts and reduces the lifespan and usefulness of the additive.

[0009] Another important limitation is the lack of complex or targeted efficacy. Currently used antimicrobial compounds work by very simple mechanisms. Their antimicrobial activity works by interacting with key metabolic enzymes and destroying key metabolic enzymes after exposure. This one-sided and simple approach limits its efficacy, duration, and uses, particularly for non-enveloped microorganisms (e.g., rhinoviruses). More complex and diverse methods are needed to create improved antimicrobial agents. Summary of the Invention

[0010] The present invention relates to bioplastics, including bioplastic materials and / or plastic additives, that, when combined with plastics, including polymers, exhibit antimicrobial properties, including antiviral properties. These properties include, but are not limited to, antiviral properties, which create an inhospitable environment for viruses, such as SARS-CoV-2. The additives of the present invention cause the plastic to create an environment inhospitable to microorganisms, including viruses, thereby preventing the growth of microorganisms and the persistence of viruses.

[0011] One form of the present invention relates to an antimicrobial polymer additive comprising a polyphenol and an organic polymer. In one advantageous form, the polyphenol is hesperidin. In an alternative advantageous form, the organic polymer is selected from chitosan (also known as chitosen), chitin, cellulose, and keratin.

[0012] In another aspect of the present invention, the aforementioned antimicrobial polymers may be added to one or more of the following polymers: chitosan and hesperidin.

[0013] In yet another alternative form, the polyphenol is 1-3% hesperidin and the organic biopolymer is 1-5% chitosan.

[0014] Without being limited to any particular antimicrobial mechanism, polyphenols (e.g., hesperidin) create surface structures (e.g., "poison tips") that are inhospitable to microbial survival. For example, when the polyphenols of the present invention are added to plastic polymers or plastics, the material possesses antimicrobial properties. Thus, the antimicrobial polymers of the present invention, including polyphenols and organic polymers, can be formulated as a surface laminate, thereby creating an antimicrobial surface.

[0015] Additionally, polyphenols and organic biopolymers can be added to coatings or formulated into plastics for medical devices such as implants and bandages.

[0016] In various alternative forms, the polyphenol or phenolic compound is hesperidin or any phenol / polyphenol including a bioflavonoid, flavonoid, tannin, coumarin, lagman, quinone, stilbene or qurcuminoid. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will now be described below with reference to the accompanying drawings.

[0018] Figure 1 is a graph comparing microbial growth of various materials according to the present invention.

[0019] Figure 2 is a graph comparing microbial growth of various materials according to the present invention.

[0020] Figure 3 is a graph comparing microbial growth of various materials according to the present invention.

[0021] Figure 4 is a graph comparing microbial growth of various materials according to the present invention.

[0022] Figure 5 is a graph comparing microbial growth of various materials according to the present invention.

[0023] Figure 6 is a graph comparing microbial growth of various materials according to the present invention.

[0024] Figure 7 is a graph comparing microbial growth of various materials according to the present invention.

[0025] Figure 8 is a graph comparing microbial growth of various materials according to the present invention. DETAILED DESCRIPTION

[0026] The bioplastics formed by incorporating the antimicrobial additives of the present invention into plastics are not suitable for microbial survival due to the unique combination of materials. The materials of the present invention are stable and have no known toxicity, making them uniquely suitable for use in various materials where antimicrobial properties are desired.

[0027] Unconstrained by any specific mechanism of antimicrobial efficacy, the combination of various additive materials creates a physical structure that proves hostile to microorganisms. At a microscopic level, the bioplastics of the present invention possess microscopic spears within the bioplastic polymer matrix. These spears or spires can be used to actively pierce bacterial (microorganism) cell walls. For example, when the organic polymer is chitosan, the chitosan spires can act as spears to penetrate the cell walls of living microorganisms.

[0028] In one form of the antimicrobial polymer, the polymer includes a polyphenol and an organic biopolymer. The organic biopolymer can be chitosan, and the polyphenol can be hesperidin. Advantageously, the chitosan forms microscopic spires within the plastic matrix of the polymer. This serves multiple functions. First, the chitosan spires are believed to be able to lyse or pierce the membranes of microorganisms. Second, the chitosan spires actively deliver the antimicrobial agent (i.e., hesperidin) to the cell walls of the microorganisms. This is an improved approach compared to the more passive reliance on migration previously known in the art. This proactive nature greatly helps increase the time it takes to kill microorganisms.

[0029] An advantage when the polyphenol is hesperidin and the organic polymer is chitosan is that both are stable, with chitosan having a melting point of 290°C and hesperidin having a melting point of 250°C. Hesperidin also has the advantage of having favorable thermal and radioprotective properties. These properties help ensure that the plastic has enhanced thermal and electromagnetic (e.g., UVA and UVB) characteristics or properties. Hesperidin also stabilizes against thermal degradation due to its antioxidant properties.

[0030] Furthermore, when the antimicrobial polymer comprises chitosan, the chitosan enhances the antimicrobial efficacy by actively dissolving the antimicrobial film and also serves as a delivery method for the antimicrobial agent hesperidin. Furthermore, hesperidin itself also functions as an antimicrobial agent.

[0031] Hesperidin inhibits microbial activity through multiple pathways. First, hesperidin inhibits biofilm formation, specifically beta-lactamase. Second, hesperidin inhibits viral replication, such as polymerases, helicases, and ATPases. A third aspect of hesperidin is its direct effect on quorum sensing by inhibiting acyl-homoserine lactase, thereby downregulating the self-induction (invasiveness) of microbial agents.

[0032] Chitosan is commercially produced by assimilating chitin, a structural element in the exoskeletons of crustaceans (such as crabs and shrimp) and in the cell walls of fungi. The chemical structure of chitin has two N-acetylglucosamine units that repeat in long chains with β-(1→4)-links, forming the following structure:

[0033]

[0034] The chemical structure of chitosan is shown below:

[0035]

[0036] The present invention will now be described with respect to experiments demonstrating the antimicrobial properties of the bioplastics and / or bioplastic additives of the present invention.

[0037] Experiment 1

[0038] Experimental data

[0039] Plastic samples containing polystyrene were created in a biosafety level 3 facility to be tested for SARS-CoV-2. Control samples were created using polystyrene as a substrate. Two "test" articles / platforms were also created. The test articles consisted of a polystyrene substrate and varying concentrations of chitosan and hesperidin. The two formulations tested are shown below; Formulation (1) = (polystyrene (97%), chitosan (4.66%), hesperidin (2.33%), Formulation (2) = (polystyrene (97%), chitosan (2%), hesperidin (1%).

[0040] Samples were sent to a Biosafety Level 3 facility for testing against SARS CoV-2. Testing procedures were designed to adhere to ISO 22196. Samples were tested in both high and low viral ranges. The high viral range was 2.5×10 5 , and the low virus range is 2.5×10 3 .

[0041] result

[0042] The control samples showed a reduction in the virus, but the test samples showed superior antiviral capabilities. Plastics coated with polystyrene / chitosan / hesperidin 93% / 4.66% / 2.33% and plastics coated with polystyrene / chitosan / hesperidin 97% / 2% / 1% reduced SARS-CoV-2 by more than 1 log(10)-fold (90% reduction) within 30 minutes and by approximately 2 log(10)-fold (99% reduction) within two hours. The antimicrobial curves of the test articles against SARS-CoV-2 continued to show a logarithmic decrease in increase after the 6-hour mark.

[0043] The results showed an incredibly rapid antimicrobial effect, with a 90% reduction in viral load within 30 minutes. It's important to note that the 30-minute mark, when samples were first analyzed, was the fastest time the lab could inoculate and then perform the assay. The consensus is that because the plastic had high antimicrobial activity within the 30-minute mark, it immediately began killing viruses.

[0044] The rapid antimicrobial efficacy demonstrated in this study is attributed to the innovative use of chitosan and hesperidin, employing an advanced and synergistic mechanism to produce an effective microscopic mechanical tool with functionality beyond that present in the one-dimensional approaches described in the current state-of-the-art.

[0045] Experiment 2

[0046] Purpose

[0047] Determining the anti-SARS-CoV-2 efficacy of bioplastic-coated plastics.

[0048] Experiment Overview

[0049] 2.5×10 5 or 2.5×10 3 TCID50 of SARS-CoV-2 was deposited on the provided coupon in a 400 μl volume. The material was transferred to an incubator maintained at 25°C and incubated for various time periods. For the 30-second treatment, the material was placed on the bench in the biosafety cabinet for the appropriate time.

[0050] The samples were incubated at room temperature for 30 seconds, 30 minutes, 2 hours, and 6 hours. The deposited virus suspension was collected and the samples were washed three times to a total volume of 1 ml. The collected suspension was diluted and various dilutions were plated onto Vero E6 cells to determine the TCID50.

[0051] All collected TCID50s were compared to TCID50s obtained from control material provided by GRDG.

[0052] Coating / surface to be tested

[0053] Control PS

[0054] PS / SLS (3%)

[0055] PS / SLS (7%)

[0056] PS / SAP (3%)

[0057] PS / SAP (7%)

[0058] PS / SAB (3%)

[0059] PS / SAB (7%)

[0060] PS / chitosan / hesperidin (93% / 4.66% / 2.33%)

[0061] PS / chitosan / hesperidin (97% / 2% / 1.0%)

[0062] Virus dose deposited on the surface

[0063] High dose - 2.5 × 10 5 TCID50

[0064] Low dose - 2.5 × 10 5 TCID50

[0065] abbreviation

[0066] SLS: Sodium Lauryl Sulfate

[0067] SAB: Strontium Aluminate Blue

[0068] SAP: Strontium aluminate violet

[0069] Chi: Chitosan

[0070] Hesp: Hesperidin

[0071] PS: polystyrene

[0072] result

[0073] The experimental data results are as follows Figure 1-8 As shown in the figure.

[0074] Experimental Discussion

[0075] After 30 seconds of exposure, no significant antiviral effect was observed in any sample. In addition, at 30 seconds, the antiviral effect was not observed in the high and low inoculum sizes (2.5×10 5 TCID50 or 2.5×10 3No significant antimicrobial effect was observed under TC1D50.

[0076] In addition, in PS / SLS and PS / SAB samples, at 30 min at high inoculum concentration (2.5×10 5 TCID50) or low inoculum (2.5×10 3 No significant antiviral effect was observed under TCID50.

[0077] In PS / Chi / Hesp ​​samples (two concentrations), a high inoculum size (2.5×10 5 A significant antiviral effect was observed at 30 minutes using the TCID50 (p<0.05). A trend toward an antiviral effect was observed using a low inoculum size, but it did not reach statistical significance (p=0.08).

[0078] In PS / SLS and PS / SAB samples, at 2 h, at high inoculum concentration (2.5 × 10 5 TCID50) or low inoculum (2.5×10 3 No significant antiviral effect was observed under TCID50.

[0079] In PS / Chi / Hesp ​​samples (two concentrations), a high inoculum size (2.5×10 5 TCID50) and low inoculum size (2.5×10 3 A significant antiviral effect was observed at 2 h (p<0.001).

[0080] In PS / SLS (3%) and PS / SAB samples, at 6 h, at high inoculum concentration (2.5×10 5 TCID50) and low inoculum size (2.5×10 3 No significant antiviral effect was observed at either TCID50 or TCID50. However, a slight decrease in viral titer was observed in the PS / SLS 7% sample (p<0.05).

[0081] In PS / Chi / Hesp ​​samples (two concentrations), a high inoculum size (2.5×10 5 TCID50) and low inoculum size (2.5×10 3 A significant antiviral effect was observed at 6 h (p<0.001).

[0082] Study limitations

[0083] These studies (six samples) were performed according to a protocol adapted from ISO 22196. Therefore, the starting SARS-CoV-2 concentration was 2.5×10 per 1” x 1” surface area in a 400 μl volume. 5 The incubation was carried out at 25°C and a relative humidity of approximately 50%.

[0084] All samples were tested at a low virus concentration (2.5×10 3 This low virus concentration does not meet ISO standards.

[0085] Plastics coated with 93% / 4.66% / 2.33% PS / chitosan / hesperidin and 97% / 2% / 1% PS / chitosan / hesperidin reduced SARS-CoV-2 by more than 1 log in 30 minutes 10 (90% reduction) and reduced by about 2 logs in 2 hours 10 (99% reduction).

[0086] Plastics coated with 7% PS / SLS reduced SARS-CoV-2 by approximately 1 log within 6 hours 10 (90%).

[0087] The following is a summary of the test conditions:

[0088] (Testing the antiviral properties of bioplastics)

[0089] Purpose

[0090] Using SARS-CoV-2 (WA1 / USA-2020) as the target microorganism, a preliminary screening was conducted to determine the potential antiviral properties of the target material (bioplastic). Further characterization was considered to use TCID 50 Titer reduction of at least one log-fold. (Based on ISO22196-SARS-CoV-2)

[0091] target

[0092] Control material / plastic or film (1, three replicates)

[0093] Coated or treated plastics or films (12 items, three replicates)

[0094] Summarize

[0095] Wipe the material with 70% ethanol for at least 3 minutes. Add 400 μl of SARS-CoV-2 (WA1 / USA-2020, 2.5 × 10 3 and 2.5×10 5PFU / ml) of culture medium was deposited on 1" x 1" of each material (three replicates) in a B2 biosafety cabinet in a certified BSL-3 laboratory. The film / material was covered with film and incubated at 25°C for 30s, 30m and 6h 24 in a humidified chamber. The material was washed three times with 300μl of culture medium and collected. 100μl of pure culture medium, 1:10, 1:100 and 1:1000 dilutions of the collected culture medium were deposited on Vero E6 cells. After incubation for 72 hours, the TCID 50 The viable virus titer was determined and calculated. The test was repeated three times. A one-log reduction in virus titer compared to the inoculum and control material in the initial screening was considered significant.

[0096] Material

[0097] 3% and 7% control materials, 60 samples. High and low concentrations tested.

[0098] SARS-CoV-2 Infectivity Assay / TCID 50 General protocol – MJN – adapted from the Sutton laboratory

[0099] 1. Prepare a 20 mg / mL stock solution in DMSO. Aliquot and store at -20°C.

[0100] 2. 24 hours before infection, seed the designated cell types in 24-well or 96-well plates and place in a CO2 incubator at 37°C.

[0101] 3. On the day of infection, prepare compound working solutions to the specified concentrations and dilute the virus stock solution to achieve the target MOI.

[0102] 4. Add virus suspension, treatment solution or a combination of both to each well at -2 hr, 0 hr or +2 hr time point as needed.

[0103] 5. Incubate the virus and cells at 37°C for 1 hour, then aspirate the virus suspension.

[0104] 6. Replace the culture medium with the treatment compound or normal culture medium and incubate the cells for the desired time.

[0105] 7. After incubation, collect the supernatant from each well and purify by centrifugation.

[0106] 8. For 24-well TCID 50 Vero E6 cells were plated 24 hours before the assay and placed in a CO2 incubator at 37°C.

[0107] 9. Continuously dilute the stock solution to 10 at a ratio of 1:10. -10 .

[0108] 10. Aspirate the old culture medium from the Vero E6 cells and add 900 μL / well of fresh culture medium containing 2% FBS.

[0109] 11. From the most diluted supernatant sample (10 -10 ) and add 100 μL of each sample to four pre-labeled Vero E6 wells. Continue adding 100 μL of each sample to the corresponding wells, starting from the most diluted sample to the most concentrated sample.

[0110] 12. Ensure that there are untreated wells for negative control.

[0111] 13. Incubate at 37°C for 72 hours, then read the cytopathic effect and calculate TCID 50 value.

[0112] Experiment 3

[0113] The antimicrobial activity of samples 1 to 8 was tested. Samples 1 to 8 are as follows:

[0114] Sample 1 – untreated paper;

[0115] Sample 2 – coated paper – strontium blue aluminate 3%;

[0116] Sample 3 – coated paper – SLS 2%;

[0117] Sample 4 – coated paper – chitosan 3%;

[0118] Sample 5 – coated paper – hesperidin 1.29%;

[0119] Sample 6 – coated paper – chitosan 1.5% / hesperidin 1%;

[0120] Sample 7 – SBSC untreated control; and

[0121] Sample 8 – Cytotoxicity optimization.

[0122] ISO 21702 specifies methods for evaluating virucidal activity on non-porous surfaces.

[0123] Each product was tested using clean testing conditions (no additional soil).

[0124]

[0125]

[0126]

[0127] ISO 21702:2019 - Measurement of antiviral activity on plastics and other non-porous surfaces

[0128] The ISO 21702 method is used to evaluate the virucidal efficacy of non-porous products. Depending on the test standard or the specific needs of the product, the test can incorporate different exposure times, soiling, virus types, and other variables. The most common test conditions use a standard method protocol that requires exposure to the test material for 24 hours based on the product's intended use. The test virus is prepared before testing, and the viral titer is then determined. The resulting inoculum is then used as an inoculum to expose the test material to the virus.

[0129] ISO 21702 Intrinsic Cytotoxicity Optimization

[0130] Antiviral testing requires the use of host cells to propagate and calculate viral concentrations. Host cells must be viable and biologically intact to allow viral infection. Successful viral infection leads to host cell replication and eventual lysis and disappearance. This process provides a means of measuring viral load. Any residual chemical residues in the test sample that may be cytotoxic to host cells may affect their biological viability and interfere with processes required by cells after viral exposure. This interference is generally considered to be inherent cytotoxicity of the test sample.

[0131] The assay was performed by incubating the test sample with a neutralizing recovery solution for 30 seconds, followed by exposure of the host cells to eight concentrations of the recovery solution. Intrinsic cytotoxicity was identified by loss of cell culture viability.

[0132] Project List

[0133] 1. Untreated paper baseline (32)

[0134] 2. Coated paper – Strontium aluminum blue 3% (24 repetitions)

[0135] 3. Coated paper – SLS 2% (24 repetitions)

[0136] 4. Coated paper – Chitosan 3% (24 replicates)

[0137] 5. Coated paper – Hesperidin 1.29% (24 replicates)

[0138] 6. Coated paper – Chitosan 1.5% / Hesperidin 1% (24 replicates)

[0139] 7.SBSC untreated control

[0140] 8. Cytotoxicity Optimization

[0141] test

[0142] Inoculum preparation

[0143] A virus suspension of known titer was prepared at a concentration of at least 10 6 TCID50 / ml. Viruses passaged more than ten times from the original seed culture were no longer used.

[0144] Experimental conditions

[0145] After inoculation, the samples were incubated at 25°C + / - 1°C (unless otherwise specified). Incubation was performed to prevent the inoculum from drying out while in contact with the test surface. After the incubation period, the virus was recovered in neutralization medium and then diluted for culture.

[0146] Virus was recovered from the test samples.

[0147] Create two time points for each test item and collect the eluate of the inoculated sample immediately after inoculation by placing the sample in a vial and adding 10 ml of the selected neutralizer, followed by vortexing to add the neutralizer solution.

[0148] A second recovery was performed after the desired incubation period (24 hr), after which the sample was placed in a vial containing 10 ml of neutralization solution and vortexed.

[0149] After neutralization of the test sample, an aliquot of the sample is recovered and used to determine the infectious titer after the corresponding incubation period.

[0150] Virus inoculum preparation

[0151] Viruses are prepared by growing a monolayer of the desired host cell culture to a density of approximately 90% confluence. The cell culture is then washed and inoculated with the prepared virus inoculum. The cells are maintained at 35 to 37°C in a 5% CO2 atmosphere, and viral replication is observed, using cytotoxicity as an indicator. Within 4 to 7 days, the cell culture is harvested and cell debris is removed by centrifugation at 1000 × g for 5 minutes. The virus culture is then titrated by serial plating according to the TCID50 method.

[0152] TCID50 cell plate

[0153] Prepare cell plates for determination of virus concentration. Inoculate virus-specific host cells in 96-well 200ul plates at a concentration of approximately 3 5 / ml. Incubate the plate with complete growth medium appropriate for the cell line. Once the cells are established, they can be inoculated with virus. Wash the plate twice with PBS, then add 100 μl of virus maintenance medium appropriate for the test virus. The cell plate is now ready for inoculation with recovered test samples.

[0154] Inoculation of test specimens

[0155] Inoculate the test article by pipetting 0.2 ml of the desired inoculum formulation onto the test article surface. Then, cover the inoculation solution with a piece of pre-cut parafilm (measuring approximately 40 mm x 40 mm - F / - 2 mm). Gently press the parafilm so that the test inoculum spreads to the edges, but not beyond the borders of the sandwich surface. Place each sample in duplicate in a sterile petri dish and then in a separate container for incubation.

[0156] Incubation of inoculated test specimens

[0157] Viral inoculum

[0158] The test substance is incubated in a water-sealed environment at a temperature of 25+ / -1°C and a relative humidity of not less than 90%.

[0159] Recovery of inoculum

[0160] Virus recovery

[0161] After the incubation period, the sample is removed from the incubator and neutralization medium is added directly to the sample and triturated 3 to 4 times. A total of 10 ml of neutralizer is added. The neutralizer is recovered and 1 ml of the solution is added to a 96-well 2 ml sample block in a column-specific manner to achieve sample processing by serial dilution. Gel filtration is used for samples that test positive for intrinsic cytotoxicity. Gel filtration is performed by recovering the neutralization solution from the sample and then passing 1 ml of the solution through a prepared 2 ml volume gel filtration bed.

[0162] Virus quantification

[0163] Viral counts were determined using a serial endpoint dilution procedure according to the TCID50 method. A sample recovery block contained 1 ml of neutralized inoculum, which was placed in the first row of an 8-row 96-well block. A 96-well block was prepared by placing 900 μl into each well of rows 2 through 8. Once the sample was added to row 1, a 1 / 10 serial dilution was performed by removing 100 μl from the first row and pipetting it into the second row, followed by trituration to mix. This process was continued for each subsequent row until a total of seven serial dilutions had been performed.

[0164] The diluted samples are then used to inoculate the prepared 96-well cell plates. For 96-well blocks, each corresponding well is added to the sample wells of two replicate 96-well cell plates. Addition is performed by aspirating 100 μl of the diluted recovered virus sample into the prepared cell plate; the total volume in the plate is 200 μl. Once all wells are transferred to the cell plate, the plate is incubated at 35°C, 5% CO2 for 2 to 6 days, depending on the characteristics of viral cytopathic effect (CPE) progression. After the incubation period, the plate is removed from the incubator, fixed and stained to identify CPE by comparison with the intact cell monolayer. The CPE / well assay is counted and the TCID50 is determined based on the representative dilution factor of the CPE for each viral sample.

[0165] Reagents

[0166] Dulbecco's modified Eagle's medium (DMEM; EM-1)

[0167] Soybean Casein Lecithin Polysorbate 80 Medium (SCDLP)

[0168] Phosphate-buffered saline (PBS)

[0169] Formaldehyde solution (3.7%)

[0170] Crystal violet (0.5%)

[0171] Fetal bovine serum

[0172] Virus maintenance medium

[0173] Trypsin

[0174] Ethylenediaminetetraacetic acid solution (EDTA)

[0175] Laboratory RO water, deionized

[0176] Test Organism (by Method) (Inventory ID / Batch #)

[0177] ISO 21702:2019 - Measurement of antiviral activity on plastics and other non-porous surfaces

[0178] Human coronavirus (OC43); VR-1558 70035458

[0179] (Beta coronavirus 1; strain: OC43)

[0180] Sample preparation

[0181] Each test substance was prepared according to the requirements of the analytical method.

[0182] For each time point, three replicates of each test sample were prepared.

[0183] If applicable, cut the specimen into pieces of approximately 50 x 50 mm. Accommodate specimen variations as needed for the standard test; any differences in specimen characteristics are noted in the report summary.

[0184] Ideally, the test specimen would be flat and non-hydrophobic, allowing the inoculum to layer over the specimen surface.

[0185] calculate

[0186] The recovered virus inoculum was subjected to endpoint dilution using serial log10 dilution factors. The TCID50 (Spearman-Karber; modified by Maramakrishnan) was used to determine the concentration of the inoculum virus based on the endpoint dilution results, thereby providing the host cell CTE. This represents the endpoint dilution (average) of the host cell monolayer that exhibited CTE.

[0187] Log10 50% endpoint dilution = -[(total number of CTE wells / total number of dilution replicates) + 0.5] × logarithmic dilution factor

[0188] R = -[total CTE / number of replicate counts per dilution) + 0.5] x log dilution factor

[0189] R = log 50% endpoint dilution

[0190] Total CTE - is the mean of the common logarithm of the number of viable viruses recovered from untreated test specimens immediately after inoculation, expressed as cells / cm 2 count

[0191] Replicate count per dilution - number of replicate wells plated at each dilution

[0192] Logarithmic Dilution Factor - This is the dilution factor used for each serial dilution (usually 10× or log10(10) = 1)

[0193] Antiviral activity value

[0194] R=U(t24)-C(t24)

[0195] R = antiviral activity value

[0196] C(t24) = the common logarithm mean of the three infectious titers of the untreated material after 24 hours

[0197] U(t24) = the common logarithm mean of the three infectious titer values ​​after a specific time (24 hr) of contact with the treated (test) sample

[0198] Statistical methods

[0199] Calculations were performed using the Spearman-Karber method using replicate data, and no additional statistical analysis was performed.

[0200] Tables 1-6 below summarize the results of this experiment:

[0201]

[0202] Table 2

[0203]

[0204] Table 3

[0205]

[0206] Although the present invention has been described above with reference to preferred embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made in these preferred embodiments without departing from the scope and spirit of the invention.

Claims

1. An antimicrobial polymer comprising: polyphenols, and Organic biopolymers.

2. The antimicrobial polymer of claim 1, wherein the polyphenol is hesperidin.

3. The antimicrobial polymer of claim 1, wherein the organic biopolymer is selected from the group consisting of chitosan, chitin, cellulose and keratin.

4. The antimicrobial polymer according to claim 3, comprising, by weight percentage: The organic biopolymer is 1-5% chitosan, and The polyphenol is 1-3% hesperidin.

5. The antimicrobial polymer of claim 1 , further comprising one or more polymers selected from the group consisting of polyethylene terephthalate (PET), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), polypropylene (PP), and polystyrene (PS).

6. The antimicrobial polymer according to claim 5, comprising, by weight percentage: The organic biopolymer is 1-5% chitosan, and The polyphenol is 1-3% hesperidin.

7. The antimicrobial polymer of claim 6, wherein the one or more polymers are present in an amount of 92% to 98% by weight.

8. The antimicrobial polymer according to claim 5, comprising, by weight percentage: 93%-97% polystyrene, The organic biopolymer is 2-5% chitosan, and The polyphenol is 1-3% hesperidin.

9. The antimicrobial polymer according to claim 5, comprising, by weight percentage: 93% polystyrene, The organic biopolymer is 4.66% chitosan, and The polyphenol was 2.33% hesperidin.

10. The antimicrobial polymer of claim 5, comprising, by weight: 97% polystyrene, The organic biopolymer is 2% chitosan, and The polyphenol was 1% hesperidin.

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